Mass spectroscopy applications speed up metabolite analysis

Two separate research teams have modified conventional mass spectroscopy techniques based on traditional matrix-assisted laser desorption/ionisation (MALDI) technology as part of their efforts to simplify the drug discovery process and speed up the analysis of metabolites.

The first is at the University of Cincinnati (UC), where scientists have shown that a new tool known as MALDI-QqQMS (matrix-assisted laser desorption ionisation-triple quadruple mass spectrometer) provides a superior means of measuring the enzyme reactions critical to drug discovery at speeds comparable to currently available high-throughput screening systems at significantly lower costs (Fig.1).

"If introduced broadly, the new generation mass spectrometry-based method we are proposing could significantly reduce the cost of running drug compound screening assays while also saving drug development teams substantial time by improving the accuracy of data collected," said Ken Greis associate professor and director of proteomics for the UC college of medicine's cancer and cell biology department.

Typical assays for enzyme screening are fluorescence and chemiluminescence-based systems. To make those assays universal, vendors have developed standard kits using specialised - and costly - reagents to identify changes in the fluorescent or chemiluminescent signals.

"There are a couple of problems with the current approach: for starters, it's an imperfect method that generates many false-positive 'hits' and for due diligence, you have to follow up on all inhibitors identified, which results in a lot of time and money wasted on false leads," says Greis.

"Reagents are very costly often ranging between 50 cents to US$1 per sample. That adds up very quickly when you're screening against a million-compound library," adds Rathore, a postdoctoral fellow in Greis' laboratory.

Greis and Rathore have developed a custom high-throughput screening method using a generalised platform. Unlike the commercially available systems that analyse by-products and coupled reactions, their system directly measures and quantifies the substrate and the end-product of the reaction.

They say using mass spectrometry to measure the mass and quantity of the product gives researchers a direct measure of the assay and more reliable compounds to explore, eliminating the chances for molecular interference common with chemiluminescence and fluorescence-based systems.

"Analytically, our mass spectrometry-based application provides superior data and also eliminates the issue of producing high numbers of false results, saving a tremendous amount of time chasing down bad leads on drug targets. And because we are using these non-tagged reagents, it only costs us 3 to 5 cents per sample to run these assays, which is a huge cost savings," adds Greis. "That can mean the difference between US$50000 and US$1million in reagent costs for a single screening project."

The approach developed by the UC group also holds appeal in that it has multiplexing capabilities-making it possible to, measure inhibitors for two or more enzymes with one pass through the compound repository. Typical assays start with one target enzyme and that is tested against an entire compound repository to look for inhibitors. Once inhibitors are identified, researchers must then follow up on each one to see if it has any validity as a drug target.

"Now instead of doing a million-dollar campaign that takes a month to run and then another million-dollar campaign that takes another month to run, we can do both at the same time while still avoiding the false-positives and false-negatives common with currently available methods," says Greis. "This is one of those disruptive technologies that could completely change the way people do this type of screening work."

Analysing metabolites

Meanwhile researchers at the Max Planck Institute for Chemical Ecology in Jena and their colleagues from the Czech Academy of Sciences in Prague have developed a new method to quickly and reliably detect metabolites, such as sugars, fatty acids, amino acids and other organic substances from plant or animal tissue samples. One drop of blood - less than one microlitre - is sufficient to identify certain blood related metabolites.

The new technique, called matrix-assisted ionisation/laser desorption (MAILD), is based on classical mass MALDI-TOF/MS spectroscopy and enables researchers to measure a large number of metabolites in biological samples, opening doors for targeted and high-throughput metabolomics. Because of its versatile applications, also in medical diagnostics, the invention is protected by patent.

In the last two decades mass spectrometry found vast applications in biology, especially for analysing of large biomolecules. MALDI, wherein bio-molecules such as proteins are co-crystallised with a chemical substance called a matrix subsequently irradiated with a laser leads to the formation of protein ions which can be analysed and detected.

However, matrices used in the MALDI technique have a substantial disadvantage: the laser beam not only forms ions from the substances of interest; it also forms low-mass ions (<500Da) originating from the matrix.

"Because of these small interfering ions we were not able to analyse small molecules that play crucial roles in the metabolism of organisms," explains Ales Svatos, head of the mass spectrometry/proteomics research group at the Max Planck Institute.

"The ions that originated from conventional matrices were like a haystack in which we wanted to find a few and important needles. Therefore the MALDI technique found only limited application in the field of metabolomics".

Instead of improving the search for the 'needles' - metabolites such as sugars, fatty acids, amino acids, and other organic acids - the scientists began to alter the matrices with which the samples were applied so that no more interfering matrix-related ions were generated. In other words they tried to remove the haystack to make the needles visible.

The researchers succeeded with the help of physical and organic chemistry, based on the Bronsted-Lowry acid-base theory, and formulated conditions for rational selection of matrices that did not generate interfering ions but provided rich mass spectra of particular kinds of metabolites in real samples.

With the new experimental protocols they called MAILD, the scientists were able to quickly and reliably determine more than 100 different analytes from single and small-sized samples. "The analysis of a very small plant leaf sample from Arabidopsis thaliana, in fact a circle area with a radius of just about 0.5mm, revealed over a hundred analyte peaks, among which 46metabolites could be identified. Interestingly, among them were eight of a total of 11 intermediates of the citric acid cycle, which is vital for most organisms," says researcher Rohit Shroff who conducted the experiments.

The new MAILD method allows measurements from diverse biological and medical materials. Apart from plant and insect samples the scientists also studied a clinical sample: they were able to determine a wide range of blood-specific organic acids in one drop of human blood, smaller than a microlitre. In medical diagnostics such measurements are still conducted with intricate methods.

If the scientists succeed in not only identifying, but also quantifying the metabolites, MAILD could develop into a fast method for medical and biological diagnostics.

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